PROJECT SUMMARY/ABSTRACT: There is substantial phylogenetic evidence that horizontal gene transfer (HGT) of host genes into large DNA viruses is a major driver of virus evolution. The status quo understanding of HGT in viruses is based exclusively on bioinformatic and phylogenetic data; however, these approaches cannot determine the molecular or evolutionary mechanisms underlying HGT. There is a critical need to develop an experimental system to directly model HGT. The long-term goal is to define how host genes are acquired by viruses and determine how these newly acquired genes evolve to provide a selective advantage to the virus. As a step towards this goal, the objective of this project is to optimize novel in vitro and in vivo experimental models to define mechanisms of HGT into poxviruses. The central hypothesis is that poxvirus HGT is facilitated through RNA intermediates. This hypothesis is based on preliminary data using our novel cell culture-based experimental model that demonstrated multiple independent long interspersed nuclear elements (LINE)-mediated HGT events into vaccinia virus (VACV). The rationale for this proposed research is that developing a robust experimental model of HGT into viruses will permit direct experimental approaches to address questions about the initial ac- quisition and subsequent evolution of captured genes that cannot be addressed by bioinformatics. For example, our preliminary data identified an unexpected evolutionary cascade that enabled transgenes to be acquired in essential genes through a process of HGT, complementation via co-infection, and subsequent recombination. The central hypothesis will be tested by three specific aims. 1) Characterize the molecular mechanisms of, and factors influencing HGT into VACV. Long-read sequencing will map the genes and surrounding genomic archi- tecture to define the mechanism(s) of HGT in cells. Cells either expressing different levels of LINE-1 or coinfected with retroviruses will be used to determine if these conditions influence HGT. 2) Analyze the evolution of hori- zontally transferred genes. HGT viruses will be serially passaged and subjected to long-read and Illumina-based deep sequencing to determine how captured genes evolve, and if there are any adaptations driven by disruption of different VACV genes due to the gene insertion event. 3) Define the mechanisms of horizontal gene transfer in vivo. A transgenic mouse line will be generated to study HGT into VACV during infection. Moreover, mouse infection studies will be performed to investigate the newly identified mechanism for sequential virus evolution following HGT into an essential gene and to assess the fitness of viruses that acquired transgenes. The proposed research is significant because it will establish and test novel experimental systems of HGT into viruses that will facilitate a detailed understanding of HGT into poxviruses that will close a fundamental gap in our knowledge of the evolution of these important viruses. This model system will open new avenues of research to directly test questions that are currently unanswerable by bioinformatic analyses such as the frequency of HGT, how the transferred genes evolve and the impact of retrovirus coinfection on HGT.